Description of microsporidia in simulids: molecular and morphological characterization of microsporidia in the larvae of Simulium pertinax Kollar (Diptera: Simuliidae)

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Major Article

INTRODUCTION

Address to:Dr.Carlos José Pereira da Cunha de Araújo-Coutinho. Laboratório de Entomologia Médica/SUCEN. Rua Cardeal Arcoverde no_{2878, 05408-003} São Paulo, SP, Brasil.

Phone: 55 11 3032-2228 e-mail: cjpcacoutinho@gmail.com Received 18 July 2014

Accepted 9 October 2014

Description of microsporidia in simulids: molecular and

morphological characterization of microsporidia in the

larvae of

Simulium pertinax

Kollar (Diptera: Simuliidae)

Isabel Maria Vicente Guedes de Carvalho

[1]

_{, Artur Trancoso Lopo de Queiroz}

[2]

_,

Rosiane Brito de Moraes

[4]

_{, Helio Benites Gil}

[3]

_{, Rafael Alves}

[5]

_,

Andréa de Barros Pinto Viviani

[6]

_{, James John Becnel}

[7]

and Carlos José Pereira da Cunha de Araujo-Coutinho

[4]

[1]. Laboratório de Parasitologia, Instituto Butantan, São Paulo, SP. [2]. Laboratório de Imunoparasitologia, Centro de Pesquisa Gonçalo Moniz, Fundação Oswaldo Cruz, Salvador, BA. [3]. Disciplina de Infectologia, Universidade Federal de São Paulo, São Paulo, SP. [4]. Laboratório de Entomologia Médica, Superintendência de Controle de Endemias, São Paulo, SP. [5]. Departamento de Medicina, Disciplina de Gastroenterologia, Universidade Federal de São Paulo, São Paulo, SP. [6]. Laboratório de Simulídeos, Superintendência de Controle de Endemias, Caraguatatuba, SP. [7]. United States Department of Agriculture, Mosquito and Fly Research Unit, Gainesville, Florida, USA.

ABSTRACT

Introduction: Microsporidia constitute the most common black ﬂ y pathogens, although the species’ diversity, seasonal occurrence

and transmission mechanisms remain poorly understood. Infections by this agent are often chronic and non-lethal, but they can cause reduced fecundity and decreased longevity. The objective of this study was to identify microsporidia infecting Simulium

(Chirostilbia) pertinax (Kollar, 1832) larvae from Caraguatatuba, State of São Paulo, Brazil, by molecular and morphological characterization. Methods: Larvae were collected at a single point in a stream in a rural area of the city and were kept under artiﬁ cial aeration until analysis. Polydispyrenia spp. infection was characterized by the presence of at least 32 mononuclear spores measuring 6.9 ± 1.0 x 5.0 ± 0.7µm in persistent sporophorous vesicles. Similarly, Amblyospora spp. were characterized by the presence of eight uninucleate spores measuring 4.5 x 3.5µm in sporophorous vesicles. Results: The molecular analysis conﬁ rmed

the presence of microsporidian DNA in the 8 samples (prevalence of 0.51%). Six samples (Brazilian larvae) were related to

Polydispyrenia simulii and Caudospora palustris reference sequences but in separate clusters. One sample was clustered with

Amblyospora spp. Edhazardia aedis was the positive control taxon. Conclusions: Samples identiﬁ ed as Polydispyrenia spp. and

Amblyospora spp. were grouped with P. simulii and Amblyospora spp., respectively, corroborating previous results. However, the 16S gene tree showed a considerable distance between the black ﬂ y-infecting Amblyospora spp. and the mosquito-infecting spp. This distance suggests that these two groups are not congeneric. Additional genomic region evaluation is necessary to obtain a coherent phylogeny for this group.

Keywords: Microsporidae. Amblyospora spp. Polydispyrenia spp.Phylogenetic analysis.

Black ﬂ ies (Diptera: Simuliidae) cause severe medical and veterinary problems worldwide. Simuliidae species are able to transmit parasites that can result in severe disease in humans and animals. In addition, their bites can cause allergic reactions and dermatitis in sensitized individuals, resulting in severe economic losses to tourism centers and negatively impacting animal production1-3. Black ﬂ y control remains a

major public health challenge. Microsporidia are unicellular, eukaryotic organisms that are obligate, intracellular parasites with public health relevance4_{. Several studies have suggested a} new classiﬁ cation for microsporidia as fungi, but Ebersberger5 stated that phylogenetic analysis did not support fungal characterization for this group.

Microsporidia are the most common black ﬂ y pathogens, although the species’ diversity, seasonal occurrence and transmission mechanisms remain poorly understood6,7_. Infections caused by this agent are often chronic and non-lethal, but they can cause sub-lethal host effects, such as reduced fecundity, decreased life span and general loss of vigor8_.

The objective of this study was to identify microsporidian species infecting Simulium (Chirostilbia) pertinax (Kollar, 1832) larvae from Caraguatatuba City, on the north coast of State of São Paulo, by molecular and morphological characterization.

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METHODS

ﬂ y bites annoy visitors and have deleterious effects on the local economy. Monitoring and controlling black ﬂ ies are essential to avoiding seasonal population outbreaks.

Sampling and biological material processing

The sampling period was from May to August 2013, and the samples were collected from a stream in Caraguatatuba City, located on the north coast of the State of São Paulo, Brazil,which has a total area of 458,097km2_{and had a population at that time} of 100,8409_{. All of the larvae were held in aerated containers} with water from the breeding site until examination. Tissues showing evidence of infection (whitish abdomens or whitish digestive tracts) were dissected in NaCl 0.9% solution, and fat bodies and adjacent tissues were removed10_{. Processed samples} were frozen in 1.5ml tubes with 30µl of diethylpyrocarbonate (DEPC)(Invitrogen® Life Technologies, Carlsbad, CA, USA). Fresh smears of fat bodies were made, ﬁ xed with methanol for 5min and stained with 10% Giemsa in 7.4 pH buffer for 20min. The slides were washed in water and dried at 25°C overnight11 for further morphological analysis of spores.

Morphological analysis

The Nis Elements F 3.0 NIKON H550S software, with phase III objective scale 100X settings, was used for spore measurement. Morphological characterization was performed according to Sprague12_.

Molecular assay

Molecular assays were performed with frozen tissues from infected larvae, and Aedes aegypti larvae infected with

Edhazardia aedis were used as positive controls.

DNA extraction

Larvae exhibiting symptoms of infection had deoxyribonucleic acid (DNA) extracted using a viral DNA kit (QIAamp® viral RNA, Qiagen, Inc, Hilden, Germany). Healthy larvae (Figure 1A)

were discarded. Tissue samples were processed with a proteinase K kit, incubated at 56°C for 2h and mixed every 20min. The supernatants were used to amplify the r16S ribosomal gene13_.

Small subunit ribosomal gene (SSUrDNA) PCR (r16S)

Polymerase chain reaction (PCR) ampliﬁ cation was performed with 18f (CAC CAG GTT GAT TCT GCC) and 1492r (GGT TAC CTT GTT ACG ACT T), according to Vossbrinck et al.14_.

The ampliﬁ cation products were visualized on 2% agarose gels, with positive and negative controls and a 100 bps ladder (Invitrogen® Life Technologies, Carlsbad, CA, USA), following electrophoresis.

Nucleotide sequencing

PCR products were purifi ed with the Illustra GFX PCR DNA and Gel Band Purifi cation Kit (GE Healthcare Limited, Little Chalfont, Buckinghamshire, UK) and were quantifi ed with 2% agarose gel ethidium bromide staining, according to the Low

DNA Mass Ladder (Invitrogen®) protocol. The products were sequenced using an ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction kit (PE Applied Biosystems), following the standard manufacturer protocols. The data were analyzed with the phred/phrap software, and the contigs were assembled with the cap3 software15_.

Phylogenetic analysis

The analyses were performed using the Seaview software16_. A phylogenetic tree was constructed, with reference sequences32-46_from_{Table 1} (supplementary ﬁ le), using the maximum likelihood method with the general time reversible (GTR) model of nucleotide substitution and gamma distribution (G) (GTR + G)17_{. The model was selected by the Modeltest} software, version 3.0.618_{, and was optimized by the Seaview} software. We calculated the bootstrap values with 1,000 replications to support the verification of branches in the topologies of the trees obtained, and bootstrap values greater than 70 were considered signiﬁ cant.

Nucleotide sequences and accession numbers

The nucleotide sequences obtained in this work were submitted to the GenBank nucleotide sequences databank under the following accession numbers GenBank: KC855552-KC855557 (L1_L6); and GenBank: KC855558 (L2).

A

B

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TABLE 1 - Sequences and accession numbers used for phylogenetic analysis.

Organism Host Geographic locale Accession number

Amblyospora bracteata Odagamia ornata Czech Republic AY09006832

Antonospora scoticae Andrena scotica USA AF024655*

Paranosema grylli Gryllus bimaculatus St. Petersburg, FL, USA AY30532533

Polydispyrenia simulii Odagamia ornata Czech Republic AY09006932

Weiseria palustris Cnephia ornithophilia USA AF132544*

Nosema algerae Anopheles stephensi Illinois, USA AF06906334

Thelohania solenopsae Solenopsis invicta USA AF03153828

Janacekia debaisieuxi Odagamia ornata USA AY09007035

Hamiltosporidium magnivora Daphnia magna Russia AJ302318.1*

Ichthyosporidium sp. Leiostomus xanthurus Not Informed L3911031

Glugea anomala Gasterosteus aculeatus Norway AF044391.136

Vavraia oncoperae Wiseana spp. New Zealand X7411237

Vavraia culicis Aedes albopictus USA AJ25296129

Endoreticulatus schubergi Lymantria dispar Switzerland L3910931

Vittaforma corneum Homo sapiens USA L3911231

Nucleospora salmonis Oncorhynchus tshawytscha Canada U7817638

Enterocytozoon bieneusi Homo sapiens USA AF02465739

Encephalitozoon cuniculi Oryctolagus cuniculus USA Z19563.140

Encephalitozoon intestinalis Homo sapiens USA U0992941

Encephalitozoon hellem Homo sapiens USA L1907042

Nosema bombycis Bombyx mori Switzerland L3911131

Vairimorpha necatrix Malacosoma americanum Not Informed Y002664

Nosema vespula Species Unknown USA U11047*

Nosema apis Apis mellifera New Zealand U97150.143

Amblyospora ferocious Psorophora ferox Argentina AY09006232

Amblyospora criniferis Aedes cernifera Argentina AY09006132

Amblyospora stimuli Diacyclops bicuspidatus USA AY09005032

Amblyospora canadensis Ochlerotatus canadensis USA AY09005632

Amblyospora cinerei Aedes cinereus USA AY09005732

Amblyospora cinerei Acanthacyclops vernalis USA AY09005932

Amblyospora cinerei Acanthacyclops vernalis USA AY09005832

Amblyospora cinerei Cyclops venustoides USA AY09006032

Amblyospora connecticus Ochlerotatus cantator USA AF025685*

Amblyospora excrucii Ochlerotatus excrucians USA AY09004332

Amblyospora stimuli Aedes stimulans USA AF02768527

Amblyospora excrucii Acanthocyclops vernalis USA AY09004432

Amblyospora khaliulini Ochlerotatus communis USA AY09004532

Amblyospora khaliulini Acanthocyclops vernalis USA AY09004632

Amblyospora khaliulini Acanthocyclops vernalis USA AY09004732

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TABLE 1 - Continuation.

Organism Host Geographic locale Accession number

Amblyospora weiseri Ochlerotatus cantans USA AY09004832

Amblyospora stictici Ochlerotatus sticticus USA AY09004932

Edhazardia aedis Aedes aegypti Thailand AF02768427

Amblyospora sp. Cyclops strenuus Czech Republic AY09005532

Amblyospora californica Culex tarsulis USA U6847344

Amblyospora sp. Culex nigripalpus USA AY09005332

Amblyospora sp. Culex salinarius USA U6847444

Amblyospora salinaria Culex salinarius USA AY32627032

Culicospora magna Culex restuans USA AY09005432

Culicospora magna Culex restuans USA AY32626932

Intrapredatorus barri Culex fuscanus Norway AY01335945

Amblyospora indicola Culex sitiens India AY09005132

Amblyospora opacita Culex territans USA AY09005232

Hyalinocysta chapmani Culiseta melanura USA AF48383746

Hyalinocysta chapmani Orthocyclops modestus USA AF48383846

Culicosporella lunata Culex pilosus USA AF02768327

Parathelohania anophelis Anopheles quadrimaculatus USA AF02768227

Parathelohania obesa Anopheles crucians USA AY09006532

Trichotuzetia guttata Cyclops vicinus Czech Republic AY32626832

Hazardia milleri Culex quinquefasciatus Argentina AY09006732

Hazardia sp. Anopheles crucians USA AY09006632

Marsoniella elegans Cyclops vicinus Czech Republic AY09004132

Gurleya vavrai Daphnia longispina Finland AF39452630

Gurleya daphniae Daphnia pulex Austria AF43932030

Larssonia obtusa Daphnia pulex Sweden AF39452730

Berwaldia schaefernai Daphnia galeata Czech Republic AY09004232

Varimorpha sp. Solenopsis richteri USA AF03153928

Amblyospora sp. Simulium sp. UK AJ25294929

USA: United States of America; FL: Florida; UK: United Kingdom.*_Unpublished.

RESULTS

A total of 1,574 S. pertinax larvae were examined. Eight larvae exhibited symptoms of microsporidian infection localized to the fat body (Figure 1B).

Morphological characterization indicated Polydispyrenia

spp. infections in 7 larvae (Figure 2A), representing 87.5% of the infected larvae. Amblyospora sp. infection was observed in one larva (12.5% of the infected larvae) (Figure 2B). The prevalence of microsporidia parasitizing larvae of S. pertinax was 0.51%.

Polydispyrenia spp. infections were characterized by the presence of at least 32 mononuclear spores contained within a persistent sporophorous vesicle, with the spores measuring 6.9 ± 1.0 x 5.0 ± 0.7µm (n = 23). Similarly, Amblyospora spp. were characterized by the presence of eight uninucleate spores contained within a sporophorous vesicle, with the spores measuring 4.5 x 3.5µm (n = 12).

The PCR products targeting the 16S region and electrophoresis agarose gel analysis conﬁ rmed the presence of microsporidian DNA in 8 samples.

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FIGURE 2 - Phase-contrast microscopy of smear slides of Simulium pertinax infected by microsporidia. Sporophorous vesicle of Polydispyrenia sp. containing 32 mononuclear spores (A). Octospores of Amblyospora spp. containing 8 uninucleate spores each (B).

A

B

[GenBank: AY090069] and Caudospora palustris [GenBank:

AF132544] reference sequences (with 100% bootstrapping). One sample (L2) was clustered with Amblyospora spp. [GenBank:

AJ252949] with 100% bootstrapping. The Edhazardia aedis

positive control (CONT+) taxon was clustered with Edhazardia aedis [GenBank: AF027684] with 100% bootstrapping.

DISCUSSION

Herein, we reported microsporidia parasitizing S. pertinax

larvae in theState of São Paulo, with a prevalence of 0.51%. Araújo-Coutinho6_{previously reported a 0.5-2.0% prevalence} of microsporidia in S. pertinax in State of Rio de Janeiro. Our study showed a similar prevalence to that previously reported by Crosskey19 in other populations of black ﬂ ies, with rates

of up to 1%. Polydispyrenia spp. were the most prevalent parasitic species in S. pertinax from Caraguatatuba/SP in this study, while Amblyospora spp. showed a higher prevalence in Rio de Janeiro6_{. This difference could be explained by the small} sample size, which prevented further analysis of the species population dynamics between S. pertinax from Rio de Janeiro and Caraguatatuba.

In this study, spores of the Polydispyrenia spp. measured 6.9 ± 1.0μm in length x 5.0 ± 0.7μm in width. Araújo-Coutinho6 reported spores of a similar size for a Polydispyrenia sp. from

S. pertinax that was ovocylindrical and measured 7.0 ± 0.6 x 4.9 ± 0.8μm. However, Castello-Branco and Andrade20_reported larger-sized spores measuring 8.3μm in length x 6.3μm in width for P. simulii from S. pertinax collectedin State ofSão Paulo,

Brazil. Sprague12_{stated that the spore dimensions were 4.5} to 5.5μm x 2.5 to 3.5μm for P. simulii with the hosts listed as

S. pertinax and S. perﬂ avum from Brazil.

In this study, for Amblyospora spp. from Caraguatatuba, the spore measurement was 4.5μm in length x 3.5μm in width, similar to that found by Araújo-Coutinho6_for_Amblyospora_spp. infecting S. pertinax in the State ofRio de Janeiro. Both of these results were similar to those from Amblyosporabracteata and

Amblyospora varians, described in black ﬂ ies in North America

and Europe21_{. According to Sprague}12

, the morphological similarity between species of microsporidia, particularly the spore measurements, makes identifi cation diffi cult, and other methods are needed for identifi cation. Such evidence indicates that spore dimension diversity is too variable; thus, molecular analysis could help in species identifi cation.

Our sample, identiﬁ ed morphologically as Polydispyrenia

spp., was grouped with the P. simulii and C. palustris clusters. This identiﬁ cation corroborated previous results22-26_regarding the phylogeny of these parasites.

The genera Parathelohania, Hazardia, Marsoniella, Gurleya, Larssonia, Berwaldia, Varimorpha, Amblyospora

and the Amblyospora sp. from S. pertinax in this study form a separate group from the main Amblyospora cluster (Figure 3). Excluding the Varimorpha sp., which wascharacterized in an ant species, Solenopsis richteri (Forel, 1909), all genera in this group are parasites of aquatics hosts27-30_.

Because the Amblyospora group is divided into two clades, corresponding to the hosts (Culex or Aedes/Ochlerotatus)28_, the aquatic group also demonstrated distinct phylogenetic characteristics according to the host. The genera that infect both

Culex quinquefasciatus (SAY, 1823)and crustaceans (Hazardia,

Marsoniella, Gurleya, Larssonia and Berwaldia) are the main members of this clade. The genera that infect anopheline mosquitoes (Parathelohania), simulids (Amblyospora spp 3 in this study) and a species of ant (Varimorpha sp.), are more closely related to the aquatic group than to the main

Amblyospora group. The Amblyospora spp. in this study were clustered with Amblyospora sp. (AJ252949) from Simulium

spp. from the Paleartic29

,; conﬁ rming the morphological and molecular similarities between these 2 species.

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FIGURE 3 - Phylogenetic tree generated for microsporidia. Unrooted tree constructed with the maximum likelihood method using the general time reversible model of nucleotide substitution and gamma distribution (GTR + G), using Seaview software. The robustness of

the phylogenetic groups was evaluated using 1,000 bootstrap replicates, and bootstrap values greater than 70 were considered signiﬁ cant.

and the main group of Amblyospora spp., which infects mosquitoes, indicating that these groups are not congeneric.

The differences between taxonomic relationships, based on phylogenetic placement and classical morphological characteristics, could probably be explained by the possibility that some of these characteristics (diplokaryon, sporophorous vesicles, and meiosis) appear to have multiple origins31_{. Thus,} molecular analysis of other genomic regions could improve the phylogenetic understanding of microsporidia. This work

contributes to the phylogenetic analysis of microsporidia because it provides two genus sequences from these parasites.

The authors declare that there is no conﬂ ict of interest.

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FINANCIAL SUPPORT

Fundação de Amparo à Pesquisa do Estado de São Paulo

(FAPESP) 2012/23947-0

REFERENCES

1. Shelley A, Hernandez L, Maia-Herzog M, Luna Dias A, Garritano D. The blackﬂ ies (Diptera: Simuliidae) of Brazil. In: Arias JR, Golovatch S, Wantzen KM, Dominguez E, editors. Aquatic biodiversity in Latin America (ABLA). Vol. 6. Pensoft:Soﬁ a-Moscow; 2010. p. 821. 2. Coscarón S, Coscarón-Arias CL. Neotropical Simuliidae (Diptera:

Insecta). In: Adis J, Arias JR, Rueda-Delgado G, Wantzen KM, editors. Aquatic biodiversity in Latim America (ABLA). Vol. 3. Pensoft: Soﬁ a-Moscow; 2007. p. 685.

3. Ribeiro do Amaral-Calvão AM, Maia-Herzog M. Adolpho Lutz's collection of black ﬂ ies (Diptera: Simuliidae), its history and importance. Hist Cienc Saude Manguinhos 2003; 10:259-271.

4. Vossbrinck CR, Maddox JB, Friedman S, Debrunner-Vossbrink BA, Woese CR. Ribosomal RNA sequence suggests microsporidia are extremely ancient eukaryotes. Nature 1987; 326: 411-414.

5. Ebersberger I, Simões RM, Kupczok A, Gube M, Kothe E, Voigt K, et al. A consistent phylogenetic backbone for the fungi. Mol Biol Evol 2012; 29:1319-1334.

6. Araújo-Coutinho CJ, Nascimento ES, Figueiro R, Becnel JJ. Seasonality and prevalence rates of microsporidia in Simulium pertinax (Diptera: Simuliidae) larvae in the region of Serra dos Órgãos, Rio de Janeiro, Brasil. J Invertebr Pathol 2004; 85:188-191.

7. Ginarte CA, Andrade CFS, Gaona JC. Larvas de simulídeos (Diptera, Simuliidae) do Centro Oeste, Sudeste e Sul do Brasil, parasitadas por microsporídeos (Protozoa) e mermitídeos (Nematoda). Ser Zool 2003; 93:325-334.

8. Castello-Branco A. Effects of Polydispyrenia simulii (Microspora; Duboscqiidae) on Development of the Gonads of Simulium pertinax (Diptera; Simuliidae). Mem Inst Oswaldo Cruz 1997; 94:421-424. 9. Online Archive of Instituto Brasileiro de Geograﬁ a e Estatística

[Internet][cited 2013 May]. Available at: http://cod.ibge.gov.br/2335A. 10. Undeen A H, Vavra J. Research Methods for Entomopathogenic

Protozoa In: Lacey LA. Manual of Techniques in Insect Pathology. Chapter 4. 1st ed. Great Britain: Academic Press; 1997. p. 115-151. 11. Becnel J J. Preparations of Entomopathogens. In: Lacey LA. Manual of

Techniques in Insect Pathology.Chapter VIII-1. 1st ed. Great Britain: Academic Press; 1997; p. 337-353.

12. Sprague V, Becnel JJ, Hazard EI. Taxonomy of Phylum Microspora. Crit Rev Microbiol 1992; 18:285-395.

13. Adler PH, Becnel JJ, Moser B. Molecular charaterization and taxonomy of a new species of Caudosporidae (Microsporidia) from black ﬂ ies (Diptera: Simuliidae), with host-derived relationships of the North American caudosporids. J Invertebr Pathol 2000; 75:133-143.

14. Vossbrinck CR, Andreadis TG, Vavra J, Debrunner-Vossbrink BA. Veriﬁ cation of intermediate hosts in the life cycles of Microsporidia by small subunit rDNA sequencing. J Eukaryot Microbiol 1998; 45:290-292. 15. Huang X, Madan A. CAP3: A DNA sequence assembly program.

Genome Res 1999; 9:868-877.

16. Gouy M, Guindon S, Gascuel O. SeaView version 4: A multiplatform graphical user interface for sequence alignment and phylogenetic tree building. Mol Biol Evol 2010; 27:221-224.

17. Waddell PJ, Steel MA. General time-reversible distances with unequal rates across sites: mixing gamma and inverse Gaussian distributions with invariant sites. Mol Phylogenet Evol 1997; 8:398-414.

18. Posada D, Crandall KA. MODELTEST: testing the model of DNA substitution. Bioinformatics 1998; 14:817-818.

19. Crosskey RW. The Natural History of Black ﬂ ies. 1st_{ed. West Sussex,} England: Wiley; 1990.

20. Castello-Branco A, Andrade CFS. Studies on Polydispyrenia simulii (Microspora; Pleistophoridae) in Simulium pertinax (Diptera; Simulidae) in Brazil. Mem Inst Oswaldo Cruz 1993; 88:167.

21. Vávra J, Undeen AH. Microsporidia (Microspora: Microsporida) from Newfoundland blakﬂ ies (Diptera: Simuliidae). Can J Zool 1981; 59:1431-1446.

22. Lom J, Nilsen F, Dykova I. Thelohania contejeani Henneguy, 1892: dimorphic life cycle and taxonomic afﬁ nities, as indicated by ultrastrutural and molecular study. Parasitol Res 2001; 87:860-872. 23. Terry RS, Smith JE, Sharpe RG, Rigaud T, Littlewood TDJ, Ironside

JE, et al. Widespread vertical transmission and associated host sex-ratio distortion within eukaryotic Phylum Microspora. Proc R Soc Lond 2004; 271:1783-1789.

24. Vossbrinck CR, Debrunner-Vossbrinck BA. Molecular Phylogeny of the Microsporidia: ecological, ultrastructural and taxonomic considerations. Folia Parasitol 2005; 52:131-142.

25. Smith JE. The ecology and evolution of microsporidian parasites. Parasitol 2009; 136:1901-1914.

26. Dong S, Shen Z, Xu L, Zhu F. Sequence and phylogenetic analisys of SSU rRNA gene of ﬁ ve Microsporidia. Curr Microbiol 2010; 60:30-37. 27. Baker MD, Vossbrinck CR, Becnel JJ, Andreadis TG. Phylogeny of

Amblyospora (Microsporida: Amblyosporidae) and related genera based on small subunit ribosomal DNA data: A possible example of host parasite cospeciation. J Invertebr Pathol 1998; 71:199-206. 28. Moser BA, Becnel JJ, Maruniak J, Patterson RS. Analysis of the

ribosomal DNA sequences of the microsporidia Thelohania and Vairimorpha of ﬁ re ants. J Invertebr Pathol 1998; 72:154-159.

29. Cheney SA, Lafranchi-Tristem NJ, Canning EU. Phylogenetic relationships of Pleistophora-like microsporidia based on small subunit ribosomal DNA sequences and implications for the source of Trachipleistophora hominis infections. J Eukaryot Microbiol 2000; 47:280-287.

30. Refardt D, Canning EU, Mathis A, Cheney SA, Lafranchi-Tristem NJ, Ebert D. Small subunit ribosomal DNA phylogeny of microsporidia that infect Daphnia (Crustacea: Cladocera). Parasitol 2002; 124:381-389.

31. Baker MD, Vossbrinck CR, Didier ES, Maddox JV, Shadduck JA. Small subunit ribosomal DNA phylogeny of various microsporidia with emphasis on AIDS related forms. J Eukaryot Microbiol 1995; 42:564-570.

32. Vossbrinck CR, Andreadis TG, Vavra J, Becnel JJ. Molecular phylogeny and evolution of mosquito parasitic Microsporidia (Microsporidia: Amblyosporidae). J Eukaryot Microbiol 2004; 51:88-95.

33. Sokolova YY, Dolgikh VV, Morzhina EV, Nassonova ES, Issi IV, Terry RS, et al. Establishment of the new genus Paranosema based on the ultrastructure and molecular phylogeny of the type species Paranosema grylli Gen. Nov., Comb. Nov. (Sokolova, Selezniov, Dolgikh, Issi 1994), from the cricket Gryllus bimaculatus. Deg J Invertebr Pathol 2003; 84:159-172.

34. Muller A, Trammer T, Chioralia G, Seitz HM, Diehl V, Franzen C. Ribosomal RNA of Nosema algerae and phylogenetic relationship to other microsporidia. Parasitol Res 2000; 86:18-23.

35. Vossbrinck CR, Andreadis TG, Vavra J, Becnel JJ. Molecular phylogeny and evolution of mosquito parasitic Microsporidia (Microsporidia: Amblyosporidae). J Eukaryot Microbiol 2004; 51:88-95.

36. Nilsen F, Endresen C, Hordvik I. Molecular phylogeny of microsporidians with particular reference to species that infect the muscles of ﬁ sh. J Eukaryot Microbiol 1998; 45:535-543.

37. Malone LA, Broadwell AH, Lindridge ET, McIvor CA, Ninham J. Ribosomal RNA genes of two microsporidia, Nosema apis and Vavraia oncoperae are very variable. J Invertebr Pathol 1994; 64:151-152. 38. Docker MF, Kent ML, Hervio DL, Khattra JS, Weiss LM, Cali A,

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39. Silva AJ, Schwartz DA, Visvesvara GS, Moura H, Slemenda SB, Pieniazek NJ. Sensitive PCR diagnosis of Infections by Enterocytozoon bieneusi (microsporidia) using primers based on the region coding for small-subunit rRNA. J Clin Microbiol 1996; 34:986-987.

40. Zhu X, Wittner M, Tanowitz HB, Cali A, Weiss LM. Nucleotide sequence of the small ribosomal RNA of Encephalitozoon Cuniculi. Nucleic Acids Res 1993; 21:1315.

41. Visvesvara GS, Silva AJ, Croppo GP, Pieniazek NJ, Leitch GJ, Ferguson D, et al. In vitro culture and serologic and molecular identiﬁ cation of Septata intestinalis isolated from urine of a patient with AIDS. J Clin Microbiol 1995; 33:930-936.

42. Visvesvara GS, Leitch GJ, Silva AJ, Croppo GP, Moura H, Wallace S, et al. Polyclonal and monoclonal antibody and PCR-ampliﬁ ed small-subunit rRNA identiﬁ cation of a microsporidian, Encephalitozoon hellem, isolated from an AIDS patient with disseminated infection. J Clin Microbiol 1994; 32:2760-2768.

43. Gatehouse HS, Malone LA. The ribosomal RNA gene region of Nosema apis (Microspora): DNA sequence for small and large subunit rRNA genes and evidence of a large tandem repeat unit size. J Invertebr Pathol 1998; 71:97-105.

44. Baker MD, Vossbrinck CR, Becnel JJ, Maddox JV. Phylogenetic position of Amblyospora Hazard & Oldacre (Microspora: Amblyosporidae) based on small subunit rRNA data and its implication for the evolution of the microsporidia. J Eukaryot Microbiol 1997; 44:220-225.

45. Nilse F, Che WJ. rDNA phylogeny of Intrapredatorus barri (Microsporida: Amblyosporidae) parasitic to Culex fuscanus Wiedemann (Diptera: Culicidae) Parasitology 2001; 122:617-623. 46. Andreadis TG, Vossbrinck CR. Life cycle, ultrastructure and molecular